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UNIVERSITY    OF    ILLINOIS    LIBRARY    AT    URBANA-CHAMPAIGN 


L161— O-1096 


BULLETIN  NO.  246 


CLOSTRIDIUM  BOTULINUM  TYPE  C 

A  PATHOGENIC  ANAEROBE  ASSOCIATED  WITH  A 
LIMBERNECK-LIKE   DISEASE   IN  CHICKENS  AND  DUCKS 

BY  ROBERT  GRAHAM  AND  I.  B.  BOUGHTON 


URBANA,  ILLINOIS,  OCTOBER,  1923 


CONTENTS  OF  BULLETIN  No.  246 

PAGE 

INTRODUCTION 3 

SPECIMENS  2770,  2771,  AND  2772 . 5 

History  5 

Clinical  Symptoms  5 

Gross  Pathology  5 

Bacteriologic  Findings    , 6 


SPECIMENS  3419  AND  3420 

History 

Gross  Pathology 

Bacteriologic  Examination 
Cultural  Characters   . 


SPECIMENS  3421,  3422,  AND  3423 16 

History  16 

Clinical  Symptoms  16 

Gross  Pathology  16 

Bacteriologic  Examination  17 

Pathogenesis 17 

SPECIMENS  3466,  3467,  AND  3468 23 

BOTULINUS  ANTITOXIN  TYPE  C 28 

THE  DEVELOPMENT  OF  TYPE  C  TOXIN  IN  SHELLED  CORN 31 

SUMMARY 32 

NOTE. — The  presence  of  Clostridittm  botulinum  type  C  in  the  digestive  tubes 
of  a  horse  and  a  steer  following  a  fatal  illness  is  noted  in  footnotes  on  pages 
28  to  30. 


CLOSTRIDIUM  BOTULINUM  TYPE  C 

A  PATHOGENIC   ANAEROBE  ASSOCIATED  WITH  A 
LIMBERNECK-LIKE   DISEASE   IN  CHICKENS  AND  DUCKS 

BY  ROBERT  GRAHAM,  CHIEF  IN  ANIMAL  PATHOLOGY  AND  HYGIENE  AND 
I.  B.  BOUGHTON,  ASSOCIATE  IN  ANIMAL  PATHOLOGY 

INTRODUCTION 

The  possible  etiologic  relation  of  botulinus  toxin  to  a  spontaneous 
syndrome  in  chickens  commonly  referred  to  as  limberneck  was  re- 
ported by  Dickson1  of  California  in  1918.  In  a  number  of  other 
states,  including  Michigan,  Washington,  and  Idaho,  avian  botulism 
has  also  occurred  in  connection  with  human  botulism  as  a  result  of 
feeding  poultry  the  unused  portions  of  the  contaminated  food.  In 
Illinois  the  sporadic  occurrence  of  avian  botulism  type  A  unassociated 
with  the  disease  in  the  human  family  has  been  suggested  in  the  results 
of  bacteriologic  findings  and  toxicogenic  tests  of  the  gastric  contents 
of  clinically  affected  chickens.  One  type  A  outbreak  following  the 
feeding  of  contaminated  garbage,  was  reported  from  Urbana  in  1921. 2 
During  the  month  of  August,  1922,  another  outbreak  which  occurred 
in  Piatt  county,  Illinois,  came  to  the  attention  of  the  authors  through 
Dr.  T.  J.  Foster,  of  Monticello.  Spoiled  over-ripe  tomatoes  were  sus- 
pected as  the  cause.  A  similar  clinical  disease  of  chickens  occurred 
at  the  White  Arrow  Farms,  Argenta,  Piatt  county,  Illinois,  in  Sep- 
tember, 1922.  Clostridium  botulinum  type  B  was  demonstrated  in  the 
intestinal  contents  and  liver  of  one  of  three  chickens  submitted  for 
examination  from  this  latter  outbreak.  This  strain,  however,  like  other 
cultures  of  type  B,  when  fed  in  massive  amounts  to  healthy  chickens, 
failed  to  induce  illness.  Definite  evidence  incriminating  type  B 
Clostridium  botulinum  or  its  toxin  as  a  primary  factor  in  poultry 
losses  has  not  been  obtained  in  cases  coming  to  the  attention  of  the 
authors. 

In  the  course  of  certain  bacteriologic  studies  of  limberneck-like 
diseases  of  poultry  resembling  botulinus  poisoning  conducted  during 
the  last  two  years,  it  became  apparent  that  neither  C.  botulinum 


1  Rockefeller  Inst.  Med.  Research,  Monograph  No.  8,  1918. 
'Journal  of  Infectious  Diseases,  28,  4,  317-322.     April,  1921. 


4  BULLETIN  No.  246  [October, 

type  A  nor  the  toxin  elaborated  by  the  type  A  organism  was  de- 
monstrable in  the  tissues  of  certain  affected  fowls.  The  limberneck- 
like  syndrome  in  spontaneously  affected  chickens,  however,  was  indis- 
tinguishable from  botulinus  poisoning.  Bacteriologic  evidence  sug- 
gesting the  character  of  the  causative  agent  was  not  obtained  in  all 
cases,  but  in  five  of  these  outbreaks  definite  and  specific  bacteriologic 
evidence  suggests  that  certain  clinical  symptoms  of  leg  weakness  and 
limberneck  occurring  in  poultry  are  associated  with  a  pathogenic 
anaerobe  which  in  preliminary  studies  appears  to  be  a  distinct  and 
separate  species  resembling,  if  not  identical  with,  C.  botulinum  type  C 
isolated  from  the  larvae  of  Lucilia  caesar  by  Bengtson1  at  the  Hygienic 
Laboratory,  Washington,  D.  C.,  and  also  the  toxicogenic  anaerobe 
isolated  from  bovine  cases  of  bulbar  paralysis  by  Seddon2  of  the  Vet- 
erinary Institute,  University  of  Melbourne.  From  three  of  these  five 
affected  flocks,  C.  botulinum  type  C  or  a  closely  related  anaerobic, 
toxin-producing,  spore-forming  rod  has  been  isolated  in  what  appears 
to  be  pure  culture.  The  cultural  and  immunologic  characters  are  dis- 
tinct from  those  of  either  type  A  or  type  B  botulinus  organisms  but 
are  analogous  to  those  of  type  C  and  the  parabotulinus  organism 
of  Seddon,  cultures  of  which  were  supplied,  respectively,  by  Dr.  Gr.  W. 
McCoy,  Director  of  the  Hygienic  Laboratory,  Washington,  D.  C.,  and 
Dr.  J.  B.  Buxton,  Professor  of  Animal  Pathology,  Cambridge  Univer- 
sity, England.  In  the  other  two  affected  flocks  the  toxic  anaerobe  was 
not  isolated  in  pure  culture,  but  the  presence  of  the  specific  type  C 
bacterial  toxin  in  impure  culture  was  demonstrated. 

In  the  affected  farm  flocks3  thus  far  observed,  type  C  avian  botul- 
ism appears  less  fatal  than  type  A.  Weakness  of  the  cervical  muscles 
(limberneck)  has  not  been  as  frequently  noted  in  outbreaks  of  type  C 
in  chickens  as  in  the  two  outbreaks  of  type  A  which  have  been 
observed,  tho  this  symptom  occurs  in  the  final  stages  of  a  highly  fatal 
type  of  both  the  spontaneous  and  the  experimentally  induced  disease. 
Healthy  chickens  under  experimental  conditions  are  less  susceptible  to 
the  filtered  and  the  unfiltered  cultures  of  type  C  than  to  those  of 
type  A.  In  fact,  some  difficulty  has  been  experienced  in  reproducing 
fatal  cases  of  the  avian  type  C  poisoning,  which  leads  to  the  belief 
that  secondary  predisposing  factors,  such  as  parasitism  or  other  bac- 
terial infections,  may  influence  the  susceptibility  of  different  chickens 
and  account  for  certain  irregularities  in  the  development  of  the  dis- 
ease under  farm  conditions.  The  potency  of  the  toxin  in  nature  may 
also  play  an  important  part. 


*U.  S.  Pub.  Health  Serv.,  Pub.  Health  Epts.,  37,  164.     1922. 
2  Jour.  Compar.  Path,  and  Ther.,  35,  147.     1922. 

*  The  disease  has  been  observed  in  one  large  poultry  establishment  in  addi- 
tion to  the  cases  referred  to  in  this  bulletin. 


1923]  CLOSTRIDIUM  BOTULINUM  TYPE  C  5 

SPECIMENS  2770,  2771,  AND  2772 

History. — During  the  month  of  July,  1921,  Drs.  Brown  and  Hinkle 
of  Decatur,  Macon  county,  Illinois,  submitted  three  chickens  to  the 
Laboratory  of  Animal  Pathology  and  Hygiene  for  bacteriologic  ex- 
amination.    A  letter  accompanying  the  specimens  stated  that  these 
fowls  had  been  ill  for  a  period  of  two  weeks.    Eight  of  the  half -grown 
chickens  in  one  pen  had  died.     The  remaining  fowls  on  the  premises 
(one  thousand)  remain- 
ed healthy.  The  first  no- 
ticeable symptom  of  ill- 
ness,   according   to   the 
owner,  was  lameness  or 
weakness  of  the  right  leg 
followed  by  incoordina- 
tion  in  walking.     Later 
both  legs  became  weak 
and  the   chickens   were 
unable    to    stand.      The 
eyes  were  partly  closed 

and  the  affected  chickens  FIG.  i.—Ascaridia  perspicilli,  found  in  chicken  2770 
appeared  dull  and  ex- 
hausted. In  the  final  stages  of  the  disease  the  comb  and  wattles  were 
a  bluish  color.  Mildly  affected  birds  recovered.  An  inquiry  into  the 
history  of  the  flock  over  a  period  of  years  failed  to  reveal  that  this  or 
a  similar  disease  had  previously  occurred  on  the  premises,  tho  symp- 
toms of  roup  had  been  recognized  by  the  owner  on  different  occasions. 
No  lesions  of  roup,  however,  were  encountered  in  the  birds  submitted 
for  examination. 

Clinical  Symptoms. — One  of  the  three  chickens  (2770)  died  on  the 
way  to  the  laboratory.  The  feathers  of  the  posterior  abdominal  region 
were  smeared  with  soft,  pasty  feces.  The  comb  and  wattles  were  dark 
in  color.  A  slimy  mucus  filled  the  mouth  and  nasal  cavities.  Chickens 
2771  and  2772  appeared  weak  and  unable  to  stand,  but  were  conscious 
and  observant  of  their  surroundings  when  aroused.  The  eyes  were 
dull  and  the  lids  were  partly  closed.  The  comb  and  wattles  were  not 
discolored. 

Gross  Pathology. — Post-mortem  changes  in  the  dead  chicken 
(2770)  masked  any  gross  lesions  which  might  have  been  present  before 
death.  In  the  other  two  chickens  (2771  and  2772)  petechial  hemor- 
rhages and  areas  of  congestion  and  catarrhal  inflammation  of  the 
mucosa  of  the  small  intestine  were  observed.  A  mass  of  Ascaridia 
perspicilli  that  partially  closed  the  lumen  was  found  in  chicken  2770 
(see  Fig.  1). 


(3 


BULLETIN  No.  246 


[October, 


Bacteriologic  Findings. — The  heart's  blood-  of  specimens  2771 
and  2772  was  seeded  on  tubes  of  slant  agar  and  found  negative  to 
Pasteurella  avium.  Rabbit  inoculation  likewise  failed  to  yield  any 
evidence  of  Pasteurella  infection.  A  composite  sample  of  the  in- 
testinal contents  of  the  three  specimens,  and  also  separate  pieces  of 
liver  tissue  from  each,  were  sown  in  glucose  pork  broth  and  brain 
media.  After  attenuating  in  a  water  bath  at  80°  C.  for  fifteen  min- 
utes in  order  to  destroy  vegetative  organisms,  the  tubes  were  incubated 
seven  to  ten  days  at  room  temperature  in  a  sealed  jar,  the  oxygen 
pressure  of  which  had  been  reduced  by  vacuum.  The  lethal  charac- 
ter of  both  the  intestinal  and  liver  cultures  (impure)  was  observed 
by  feeding  .5  to  Ice.  to  guinea  pigs.  Death  was  also  induced  in  other 
guinea  pigs  by  feeding  similar  amounts  of  the  sterile  filtrate.  The 
symptoms  observed  preceding  death  resembled  those  of  botulinus 
poisoning. 

Neither  A  nor  B  botulinus  antitoxins  injected  subcutaneously  in 
guinea  pigs  at  the  time  they  were  fed  toxic  cultures  2770,  2771,  and 
2772,  afforded  any  protection  (see  Tables  1,  2,  and  3).  These  anti- 
toxins also  failed  to  protect  guinea  pigs  when  injected  simultaneously 
twenty-four  hours  previous  to  their  being  fed  culture  filtrates  2770, 
2771,  and  2772,  and  at  the  time  of  feeding,  altho  a  non-specific 


TABLE  1. — NON-PROTECTIVE  CHARACTER  OF  BOTULINUS  ANTITOXINS  A  AND  B 
AGAINST  CULTURE  FILTRATE  2770 


Identification 

Treatment, 

September  15,  1921 

Results 

Guinea 
pig 

No. 

Weight 

963 

300  gms. 

.  5  cc.   culture 
mouth 

filtrate 

2770 

by 

tOO  units  type  A  botulinus  anti- 
toxin subcutaneously 

Died  9/17/21 

489 

300  gms. 

.  5  cc.   culture 
mouth 

filtrate 

2770 

by 

100  units  type  B  botulinus  anti- 
toxin subcutaneously 

Died  9/17/21 

544 

300  gms- 

.  5  cc.   culture 
mouth 

filtrate 

2770 

by 

Control 

Died  9/16/21 

TABLE  2. — NON-PROTECTIVE  CHARACTER  OF  BOTULINUS  ANTITOXINS  A  AND  B 
AGAINST  CULTURE  FILTRATE  2771 


Identification 

Treatment, 

September  9,  1921 

Results 

Guinea 
pig 

No. 

Weight 

987 

300  gms. 

.5  cc.  culture 
mouth 

filtrate 

2771 

by 

100  units  type  A  botulinus  anti- 
toxin subcutaneously 

Died  9/10/21 

979 

300  gms. 

.5  cc.  culture 
mouth 

nitrate 

2771 

by 

100  units  type  B  botulinus  anti- 
toxin subcutaneously 

Died  9/10/21 

485 

300  gms. 

.5  cc.  culture 
mouth 

filtrate 

2771 

by 

Control 

Died  9/10/21 

1923} 


CLOSTRIDIUM  BOTULINUM  TYPE  C 


TABLE  3. — NON-PROTECTIVE  CHARACTER  OF  BOTULINUS  ANTITOXINS  A  AND  B 

AGAIMST  CULTURE  FILTRATE  2772 


Identification 

Treatment,  September  15,  1921 

Results 

Guinea 
pig 
No. 

Weight 

984 

300  gms. 

.5  cc.  culture 
mouth 

filtrate 

2772  by 

100  units  type  A  botulinus  anti- 
toxin subcutaneously 

Died  9/19/21 

543 

300  gms. 

.5  co.  cultuie 
mcutn 

filtrate 

2772  by 

100  units  type  B  botulinus  anti- 
toxin subcutaneously 

Died  9/16/21 

072 

300  gms. 

.5  cc.  culture 
mouth 

filtrate 

2772  by 

Control 

Died  9/16/21 

and  incomplete  protection  was  occasionally  observed.  These  findings 
were  carefully  checked  by  demonstrating  the  protective  character  of 
the  antitoxins  against  known  A  and  B  filtered  cultures  of  the  botulinus 
organism.  The  preliminary  results  of  typing  tests  were  conflicting 
only  when  less  than  lethal  amounts  of  the  culture  or  culture  filtrate 
were  used. 

Stained  smears  of  seven-  to  ten-day-old  cultures  from  the  liver  tis- 
sues of  specimens  2770,  2771,  and  2772  showed  at  least  two  types  of 
anaerobes — a  facultative  anaerobic,  Gram-positive  diplococcus  and  a 
few  terminal  spore-bearing,  Gram-negative  rods.  The  diplococcus 
proved  to  be  non-pathogenic.  An  effort  to  purify  and  cultivate  the 
spore-bearing  anaerobe  by  heating  and  seeding  in  glucose  agar  failed. 
While  the  cultural  characters  of  this  organism  were  not  determined, 
preliminary  immunologic  tests  suggested  the  presence  of  a  toxin- 
producing  anaerobe  which  was  capable  of  inducing  the  botulism 
syndrome  but  which  was  apparently  neither  A  nor  B  type  of  (7. 
botulinum.  The  methods  used  and  the  media  in  which  subcultures 
were  seeded,  while  satisfactory  for  the  isolation  of  types  A  and  B, 
did  not  seem  to  meet  the  requirements  of  the  toxin-producing  rod 
encountered. 

Bacteriologic  examination  of  soil  samples  obtained  from  the 
chicken  yards  from  which  specimens  2770,  2771,  and  2772  had  been 
received  failed  to  show  the  presence  of  a  pathogenic  anaerobe.  In 
view  of  the  positive  results  obtained  later  from  other  soils,  however, 
the  authors  are  inclined  to  think  that  these  negative  results  may  be 
attributed  to  the  media  employed,  which  have  since  appeared  to  be 
unsatisfactory  for  the  isolation  of  this  organism. 

During  the  year  following  the  discontinuing  of  the  above  bacterio- 
logic  study,  the  intestinal  contents  and  internal  organs  of  several 
suspected  chickens  submitted  from  different  flocks  for  diagnosis  were 
cultured  for  the  presence  of  toxic  anaerobes,  but  always  with  negative 
results. 


8  BULLETIN  No.  246  [October, 

SPECIMENS  3419  AND  3420 

History. — One  hen  (3419)  and  one  rooster  (3420)  were  received 
from  a  sick  flock  belonging  to  Mr.  John  Thomen,  GTreenup,  Cumber- 
land county,  Illinois,  on  July  1,  1922.  The  hen  appeared  dull  and 
droopy,  while  the  rooster  was  moribund  and  suffered  from  a  severe 
diarrhea.  Neither  showed  symptoms  of  limberneck.  A  complete 
history  of  the  disease  in  the  flock  was  not  obtained,  tho  a  letter  ad- 
vised that  the  flock  had  been  fed  the  carcasses  of  chickens  that  had 
died  of  limberneck.  It  was  estimated  by  the  owner  several  months 
later  that  sixty  birds  had  died  of  the  disease. 

Gross  Pathology. — Intestinal  parasitism  was  encountered  in  speci- 
men 3420  at  autopsy,  about  twenty  tapeworms  and  numerous 
Heterakis  papillosae  being  found  in  the  ceca.  Diffuse  areas  of 
catarrhal  enteritis  were  found  in  the  duodenum.  Specimen  3419 
was  held  for  observation.  It  appeared  normal  and  active  after  the 
second  day. 

Bacteriologic  Examination. — Aerobic  cultures  from  the  heart's 
blood  of  specimen  3420  were  negative,  but  the  anaerobic  cultures  in 
meat-mash  medium  from  the  intestinal  contents  and  liver  proved  lethal 
to  guinea  pigs.  The  symptoms  induced  resembled  botulism,  but  the 
course  of  the  disease  was  in  no  way  modified  by  the  subcutaneous  in- 
jection of  botulinus  antitoxins  of  either  type  A  or  type  B.  Botulinus 
antitoxin  type  C,  however,  apparently  provided  a  specific  protection 
(see  Table  4).  Colonies  in  deep  glucose-agar  cultures  as  well  as 

TABLE  4. — PROTECTIVE  CHARACTER  OF  BOTULINUS  ANTITOXINS  TYPE  C 
AGAINST  CULTURE  FILTRATE  3420 


Identification 

Treatment, 

July  25,  1922 

Results 

Guinea 
pig 

No. 

Weight 

659 

300  gms. 

.5  cc.  culture 
mouth 

filtrate  3420 

by 

1  cc.  type  C  botulinus  antitoxin 
2961  subcutaneously 

Remained  healthy 
Released  8/25/22 

799 

300  gms. 

.5  cc.  culture 
mouth 

filtrate  3420 

bar 

Control 

Died  7/26/22 

•Furnished   by   Dr.   G.   W.    McCoy,   Director,   Hygienic   Laboratory,   Washington,    D.   C. 

liver-agar    anaerobic    plates    yielded    a    pathogenic    spore-bearing 
anaerobe  in  pure  culture. 

Cultural  Characters. — The  following  description  of  the  cultural 
characters  of  pathogenic  anaerobe  3420  is  based  upon  a  study  of 
single-colony  subcultures  in  glucose  agar.  Non-toxic  subcolony  cul- 
tures from  single  colonies  have  been  encountered,  but  a  study  of  the 
morphology  of  the  organism  together  with  the  growth  characteristics 


1923}  CLOSTRIDIUM  BOTULINUM  TYPE  C  9 

leads  to  the  belief  that  a  pure  culture  may  contain  not  only  non-toxic 
cells,  as  mentioned  by  Bengtson,1  but  also  colonies  which  on  trans- 
plantation fail  to  elaborate  toxin.  The  reaction  of  media  or  conditions 
of  growth,  such  as  temperature  and  oxygen  requirements,  may  possibly 
influence  the  number  of  toxic  cells  in  a  culture,  since  serum  agar  or 
glucose  agar  in  inverted  petri-dish  plate  cultures  incubated  at  37°  C. 
yield  a  relatively  high  number  of  non-toxic  colonies.  The  appear- 
ance of  the  colonies  on  subculturing  in  glucose-agar  tubes,  the  char- 
acter of  growth  of  subcultures  in  meat  media  as  well  as  the  morphology 
and  staining  characters  do  not  serve  to  distinguish  toxic  from  non- 
toxic  cultures  obtained  from  single  colonies. 

Conditions  of  Growth. — Clostridium  botulinum  type  C  is  a  strict 
anaerobe.  It  grows  well  in  media  containing  meat  (beef  muscle,  pork 
muscle,  liver)  in  unsealed  tubes.  Scant  growth  on  solid  media  may 
be  obtained  in  an  atmosphere  of  hydrogen,  providing  a  heavy  inoculum 
is  used.  Satisfactory  anaerobiosis  may  be  secured  in  glucose-agar 
plate  cultures  by  sealing  the  inverted  petri  dish  in  the  lid.  More 
consistent  growth  has  been  obtained  in  subcultures  from  glucose  agar 
when  heavily  inoculated.  Growth  occurs  at  22°  C.  but  more  rapidly 
at  37°  C. 

Morphology. — The  organism  is  a  clostridium  with  rounded  ends 
about  .9  micron  wide  and  5  to  8  microns  long  (see  Figs.  2  and  3). 
In  old  meat-mash  cultures  longer  and  more  slender  forms  have  been 
observed.  Cultures  in  meat  mash  forty-eight  hours  old  incubated 
at  37°  C.  frequently  show  terminal  spores.  Often  two  or  more 
organisms  are  observed  in  short  chains,  while  in  old  cultures  a  few 
long  chains  may  extend  across  the  field.  Intracellular  or  extracellular 
spores  are  present  depending  on  the  age  of  the  culture.  The  latter 
are  numerous  in  old  meat-mash  cultures.  The  spores  are  oval  in 
shape,  bulging  the  cell  wall,  and  occur  singly  at  the  end  of  the  vege- 
tative cell.  They  are  not  heat  resistant.  An  exposure  to  80°  C.  for 
five  minutes  is  generally  sufficient  to  render  them  inert. 

Motility. — In  examining  cultures  twenty-four  to  forty-eight  hours 
old  in  cover-glass  films,  no  motility  has  been  observed. 

Staining. — The  Clostridium  stains  readily  with  ordinary  aniline 
dyes.  In  young  cultures  the  organism  is  Gram-positive.  This  char- 
acter, however,  is  not  constant  as  Gram-negative  cells  appear  after 
three  or  four  days.  Many  Gram-negative  staining  organisms  may 
contain  Gram-positive  granules  in  cultures  five  days  old. 

Plate  Cultures. — In  petri-dish  cultures  made  anaerobic  according 
to  Krumwiede  and  Pratt 's  method,  scant  growth  may  be  obtained  in 
glucose  and  in  liver  agar  (see  Fig.  4).  In  the  former  medium,  gas  in 
small  amounts  is  produced.  The  growth  is  translucent,  whitish  in 
color,  and  made  up  of  cottony,  loosely  constructed  colonies.  In  older 


'U.  S.  Pub.  Health  Serv.,  Pub.  Health  Rpts.,  37,  2252.     1922. 


10 


BULLETIN  No.  246 


[October, 


FIG.  2. — SPOKES  OF  C.  botulinum  TYPE  C  (3420). 
AN  OLD  CENTEIFUGALIZED  MEAT-MASH  CULTURE,   x  1200 


FIG.  3. — C.  botulirmm  TYPE  C  (3420).    A  CENTRIFUGALIZED 
MEAT-MASH  CULTURE  FORTY-EIGHT  HOURS  OLD.   x  1200 


1923}  CLOSTRIDIUM  BOTULINUM  TYPE  0  \\ 

plate  cultures  the  growth  becomes  more  compact  and  opaque  with  a 
faint  resemblance  to  frosted  glass.  In  liver-agar  plates  the  character 
of  growth  is  similar,  but  disrupted  and  broken  by  gas  production.  The 
distinctness  of  the  growth  disappears  in  old  plate  cultures,  and  sub- 
cultures from  plates  are  obtained  with  difficulty.  Serum-agar  plate 
cultures  incubated  in  hydrogen  atmosphere  (Brown  jar)  show  a  faint 
growth  at  points  heavily  inoculated.  The  edges  of  the  colonies  are 
irregular  and  have  many  short,  fine,  spiney  projections  extending  out 
irregularly.  Stained  smears  of  plate  colonies  fail  to  show  spores.  A 
larger  percentage  of  non-toxic  subcultures  have  been  obtained  from 
colonies  in  plates  than  from  colonies  in  glucose-agar  shake  cultures. 

Meat  Medium. — In  meat-mash  medium  of  Bengtson  (one  part 
finely  chopped  lean  meat  to  two  parts  water)  the  most  favorable 
growth  has  been  obtained.  Gas  production  occurs  in  twenty-four  to 
forty-eight  hours  at  37°  C.  The  meat  remains  undigested,  and  the 
liquor  relatively  clear.  The  meat  is  not  discolored  during  growth. 
Gas  production  is  generally  very  marked.  The  bubbles  are  large  and 
do  not  collect  on  the  surface  of  the  liquid  but  may  be  seen  in  the 
meat  along  the  sides  of  the  tubes  (see  Fig.  5,  A).  After  six  to  eight 
days,  gas  production  generally  subsides,  and  the  inoculated  tube 
appears  clear.  Spore  formation  occurs  in  meat  mash  in  thirty-six 
hours  at  37°  C.  A  slight  odor  is  noticeable  in  actively  growing  cul- 
tures, but  it  does  not  resemble  the  butyric  acid  odor  of  either  the 
A  or  the  B  botulinus  organism. 

Liver  Medium. — In  liver  medium  made  in  the  same  proportion  as 
the  meat  medium  employed  by  Bengtson,  the  growth  is  similar  to 
that  in  meat-mash  cultures  and  resembles  the  growth  of  B.  novyii  and 
certain  non-pathogenic,  sporulating  anaerobes. 

Glucose  Broth. — In  glucose  broth  a  suspended  flocculent  growth 
occurs  at  37°  C.  The  flocculi  settle  to  the  bottom  of  the  tube  in  a 
white,  cottony  mass  or  collect  as  a  fine  sediment  along  the  side  of  the 
tube,  leaving  the  broth  clear.  Acid  is  produced  but  no  gas. 

Glucose  Agar. — In  2-percent  glucose-agar  deep  tubes  at  37°  C. 
growth  occurs  two  or  more  centimeters  below  the  surface.  The  colonies 
are  translucent,  white,  and  irregular  in  shape,  with  fine,  cottony, 
fiber-like  projections.  The  maximum  growth  is  generally  attained  on 
the  fourth  or  fifth  day,  after  which  the  distinctness  of  the  growth 
decreases.  Similar  types  of  colonies  occur  in  liver  agar  with  mild 
gas  production  (see  Fig.  5,  B). 

One-tenth  Percent  Agar. — In  .1  percent  agar,  well  defined  sepa- 
rated colonies  have  not  been  observed  at  37°  C.  Two  or  more  centi- 


12 


BULLETIN  No.  246 


[October, 


meters  below  the  surface  the  inoculated  tubes  show  a  uniform 
cloudiness. 

MUk. — In  milk,  acid  is  produced. 

The  cultural  characters  of  value  in  differentiating  Clostridium 
botulinum  types  A  and  B  from  type  C  or  the  parabotulinus  organism 
of  Seddon  are  summarized  in  Table  5. 

TABLE  5. — CULTURAL   CHARACTERS  DIFFERENTIATING   CLOSTRIDIUM  BOTDLINUM 
TYPES  A  AND  B  FROM  TYPE  C  AND  PARABOTULINUS  OF  SEDDON 


Clostridium  botulinum 
types  A  and  B 

Clostridium  botulinum  type  C  or 
parabotulinus  'Seddonl 

Glucose  agar  

Gas 

No  gas 

Glucose  agar  

Disc  colonies 

Branching  colonies 

Glucose  broth  

Even  cloudiness 

Flocculent  growth 

Glucose  broth  

Acid  and  gas 

Acid 

Milk  

No  change 

Acid 

Motility             

Motile  under  cover  glass 

Non-motile  under  cover  glass 

Resistant  to  heat 

Non-resistant  to  heat 

Pafho genesis. — The  non-lethal  character  of  small  amounts  of  the 
type  C  nitrate  3420  given  by  the  mouth  is  indicated  in  the  results 
obtained  in  chickens,  horses,  pigs,  calves,  and  rabbits,  which  are 
reported  in  Table  6.  Larger  amounts,  however,  when  fed  to  these 
animals  in  some  cases  produced  illness.  In  chickens  the  symptoms 
experimentally  induced  resemble  clinically  those  of  the  disease 
occurring  under  farm  conditions. 

The  fact  that  small  amounts  of  the  culture  or  culture  nitrate  may 
be  consumed  by  fowls  as  well  as  by  other  domestic  animals  without 
producing  illness  suggests  that  either  unusually  large  amounts  are 
necessary  to  produce  the  disease  or  the  invasive  character  or  potency 
of  the  toxin  in  nature  is  greater  than  that  of  the  toxin  produced  in 
artificial  cultures.  When  subcutaneously  injected,  both  the  unfiltered 
and  the  filtered  culture  are  more  quickly  fatal  to  horses,  cattle,  and 
sheep  than  when  given  in  the  feed,  tho  some  chickens  have  withstood 
single  subcutaneous  injections  of  3  cc.  and  4.5  cc.,  which  represented 
.05  cc.  and  .01  cc.,  respectively,  per  gram  live  weight. 

Baeteriologic  examination  of  soil  samples  obtained  during  the 
month  of  January,  1923,  from  the  chicken  yard  from  which  specimen 
3420  was  received,  yielded  positive  evidence  of  C.  botulinum  type  C. 
The  relation  of  the  soil  contamination  to  the  disease  occurring  in  the 
fowls,  however,  is  not  known. 


B 


FIG.  4. — C.  botulinum  TYPE  C  (3420)  :    INVERTED  PETRI-DISH  CULTURES 
(A)  IN  GLUCOSE  AGAR;    (B)  IN  LIVER  AGAR 


FIG.  5. — C.  botulinum  TYPE  C  (3420) :   TUBE  CULTURES  (A)  IN 
MKAT  MASH;    (B)  ix  LIVER  AGAB 


1923] 


CLOSTRTDIUM:  BOTULINUM  TYPE  C 


15 


TABLE  6. — EFFECT  OF  SMALL  AMOUNTS  OF  TTPE  C  CULTURE  FILTRATE  3420 

GIVEN  BY  MOUTH 


Identification 

Treatment,  July  26,  1922 
Filtrate  3420  by  mouth 

Results 

No. 

Species 

Weight 

3518 
866 

Chicken 

1050  gms. 

1  cc. 

Remained  healthy 
Released  8/11/22 

3519 
862 

Chicken 

1130  gms. 

2  cc. 

Remained  healthy 
Released  8/11/22 

3520 
852 

Chicken 

1310  gms. 

4  cc. 

Remained  healthy 
Released  8/11/22 

3521 

872 

Chicken 

1300  gms. 

5  cc.' 

Remained  healthy 
Released  8/11/22 

3522 
768 

Chicken 

610  gms. 

8  cc. 

Remained  healthy 
Released  8/11/22 

3523 
856 

Chicken 

1170  gms. 

10  cc. 

Remained  healthy 
Released  8/11/22 

3525 
18B 

Horse 

1550  Ibs. 

10  cc. 

Remained  healthy 
Released  8/11/22 

3526 
21A 

Horse 

1500  Ibs. 

20  cc. 

Remained  healthy 
Released  8/1  1/22 

3527 
13DJ 

Pig 

60  Ibs. 

1  cc. 

Remained  healthy 
Released  8/11/22 

3528 
30H 

Pig 

50  Ibs. 

2  cc. 

Remained  healthy 
Released  8/1  1/22 

3529 
10H 

Pig 

40  Ibs. 

3  cc. 

Remained  healthy 
Released  8/11/22 

3530 
2DJ 

Pig 

251be. 

6  cc. 

Remained  healthy 
Released  8/11/22 

3531 
99DJ 

Pig 

25  Ibs. 

12  cc. 

Remained  healthy 
Released  8/11/22 

3532 
5H 

Pig 

30  Ibs. 

24  cc. 

Remained  healthy 
Released  8/11/22 

3533 
184 

Calf 

400  Ibs. 

5  cc. 

Remained  healthy 
Released  8/11/22 

3534 
187 

Calf 

400  Ibs. 

10  cc. 

Remained  healthy 
Released  8/11/22 

3535 
188 

Calf 

400  Ibs. 

20  cc. 

Remained  healthy 
Released  8/1  1/22 

3536 
820 

Guinea  pig 

280  gms. 

.5  cc. 

Died  7/28/22,  7  a.m. 

3537 
684 

Guinea  pig 

330  gms. 

1  cc. 

Died  7/28/22,  7  a.m. 

3538 
721 

Guinea  pig 

370  gms. 

1.5  cc. 

Died  7/28/22,  7  a.m. 

3540 
622 

Rabbit 

2830  gms. 

2  cc. 

Transitory  illness;  re- 
covered 
Released  8/11/22 

3541 
613 

Rabbit 

2900  gms. 

• 
4  cc. 

Remained  healthy 
Released  8/1  1/22 

3.542 
612 

Rabbit 

2600  gms. 

8cc. 

Died  7/28/22,  10  a.m. 

16 


BULLETIN  No.  246 


[October, 


SPECIMENS  3421,  3422,  AND  3423 

History. — Two  chickens  and  one  duck  from  a  farm  flock  of  two 
hundred  fowls  were  submitted  by  Mr.  A.  E.  Davidson  of  Mt.  Sterling, 
Brown  county,  Illinois,  on  July  12,  1922,  for  bacteriologic  examina- 
tion. From  the  history  of  the  outbreak,  given  by  the  owner  of  the 
flock,  it  was  learned  that  the  disease  had  appeared  simultaneously 
with  the  feeding  of  a  certain  lot  of  wheat.  Twelve  mature  hens,  four- 
teen half-grown  pullets,  and  one  duck  had  died,  and  several  birds 

were  dull  and  droopy. 
Some  chickens  of  the  flock 
were  normal  in  appearance 
but  were  unable  to  walk. 
The  ground  of  the  chicken 
lot  was  strewn  with  feath- 
ers that  had  fallen  from  the 
affected  fowls.  A  similar 
disease,  according  to  the 
observation  of  the  owner, 
had  occurred  on  these  prem- 
ises the  preceding  summer. 
A  part  of  the  chicken  range 
on  this  farm  was  bottomland  and  overflowed  each  spring. 

Clinical  Symptoms. — Chicken  3421  died  en  route  to  the  laboratory. 
Chicken  3422  showed  symptoms  of  leg  weakness,  remaining  in  a  nor- 
mal sitting  posture  (see  Fig.  6).  Duck  3423  was  moribund  (see 
Fig.  7).  Apparently  it  could  not  raise  its  head,  but  frequently 
moved  it  from  side  to  side  on  the  ground.  The  eyes  were  closed. 
When  aroused  the  duck  responded  only  by  quacking  faintly. 
Respirations  were  slow,  deep,  and  regular.  Diarrhea  was  a  promi- 
nent symptom.  The  feathers  came  out  when  the  duck  was  handled. 


Fig.  6. — Chicken  3422,  apparently  healthy  but 
unable  to  stand 


FIG.  7. — DUCK  3423  IN  A  MORIBUND  CONDITION 


Gross  Pathology. — In  the  crop  of  chicken  3421  fully  fifty  fly  larvae 
were  found.     A  few  Heterakis  perspicilli  were  noted  in  the  small 


1923]  CLOSTRIDIUM  BOTULINUM  TYPE  C  17 

intestine.  Aside  from  small  areas  of  hyperemia  in  the  intestinal  mu- 
cosa,  no  lesions  were  found.  The  intestinal  contents  of  the  three  speci- 
mens were  extracted  for  thirty  minutes  in  saline  solution  and  1  cc. 
of  the  solution  was  then  fed  to  guinea  pigs.  Symptoms  of  botulism 
and  death  followed  in  twenty-four  hours  or  less. 

Bacteriologic  Examination. — Aerobic  cultures  of  the  internal 
organs  failed  to  reveal  evidence  of  Pasteurella  avium.  Anae- 
robic cultures  from  the  intestinal  contents  and  liver  of  the  three 
specimens  proved  lethal  when  fed  to  guinea  pigs.  A  faintly  Gram- 
positive,  spore-forming  rod,  which  culturally  resembled  anaerobe 
3420,  was  isolated  from  the  liver  of  chicken  3421  by  the  deep-tube 
method.  The  feeding  of  culture  3421,  as  well  as  the  culture  nitrate, 
to  guinea  pigs  produced  a  fatal  botulinus  syndrome.  No  protection 
was  obtained  by  injections  of  either  A  or  B  botulinus  antitoxins. 
Specific  protection,  however,  was  apparently  afforded  by  botulinus 
antitoxin  type  C,  received  from  the  Hygienic  Laboratory,  Washington, 
D.  C.  (see  Table  7). 

TABLE  7. — PROTECTIVE  CHARACTER  OF  BOTULINUS  ANTITOXIN  TYPE  C 
AGAINST  CULTURE  FILTRATE  3421 


Identification 

Treatment,  July  25,  1922 

Results 

Guinea 
pig 

No. 

Weight 

671 

300  gms. 

.5  cc.  culture  filtn 
mouth 

ite  3421  by 

1  cc.  type  C  botulinus  antitoxin 
296  l  subcutaneously 

Remained  healthy 
Released  8/25/22 

654 

300  gms. 

.5  cc.  culture  filtrate  3421  by 
mouth 

Control 

Died  7/27/22 

"Furnished  by  Dr.  G.  W.  McCoy,  Director,  Hygienic  Laboratory,  Washington,  D.  C. 

Pathogenesis. — The  subcutaneous  injection  of  .01  cc.  to  .001  cc.  of 
culture  and  culture  nitrate  3421  proved  fatal  to  guinea  pigs  and 
rabbits,  as  did  also  the  feeding  of  .5  cc.  to  guinea  pigs.  The  feeding 
of  10  cc.  of  culture  and  culture  nitrate  to  horses,  calves,  and  pigs  for 
ten  consecutive  days  was  without  noticeable  effect  (see  Table  8).  A 
chicken  (3667)  fed  3  cc.  of  the  culture  daily  for  ten  consecutive 
days  succumbed,  but  another  chicken  (3668)  fed  the  culture  nitrate 
in  similar  amounts  each  day  showed  no  ill  effect.  Two  dogs,  one 
consuming  5  cc.  of  the  culture  and  the  other  5  cc.  of  the  culture 
nitrate,  for  ten  consecutive  days,  remained  healthy.  The  toxic  prop- 
erty of  the  culture  nitrate  was  determined  at  the  beginning  and  the 
termination  of  t,he  feeding  period  by  feeding  .5  cc.  to  guinea  pigs. 

Massive  amounts  of  the  culture  have  been  fed  to  chickens,  horses, 
cattle,  and  pigs.  Some  chickens  have  displayed  a  marked  resistance 
to  the  culture  and  culture  filtrate  in  the  feed,  consuming  as  high 
as  one  gram  per  thirty  grams  live  weight,  or  approximately  3  percent 


18 


BULLETIN  No.  246 


[October, 


Results 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Marked  leg  weak- 
ness 8/16 
Died  8/19/22 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Remained  healthy 
Released  8/22/22 

Died  8/8/22 

Died  8/9/22 

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Animal 

Horse  A,  No.  21 

Horse  B,  No.  18 

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female 

Dog;  black  and 
white  male 

Guinea  pig,  No. 
606 

Guinea  pig,  No. 
602 

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CLOSTRIDIUM  BOTULINUM:  TYPE  C 


19 


TABLE  9. — RESISTANCE  OP  CHICKENS  TO  TYPE  C  CULTURE  FILTRATE  3421 

GIVEN  BY  MOUTH 


Identification 

Treatment,  October  31,  1922 

Results 

Chicken  No. 

Weight 

455 

1860  gms. 

7.7  cc.1  culture  filtrate  3421  by  mouth 

Remained  healthy 
Released  11/14/22 

456 

2530  gms. 

21  cc.2  culture  filtrate  3421  by  mouth 

Remained  healthy 
Released  11/14/22 

457 

2110  gms. 

35  cc.*  culture  filtrate  3421  by  mouth 

Remained  healthy 
Released  11/14/22 

458 

2180  gms. 

36.6  cc.»  culture  filtrate  3421  by  mouth 

Remained  healthy 
Released  11/14/22 

459 

2000  gms. 

66  cc.4  culture  filtrate  3421  by  mouth 

Remained  healthy 
Released  11/14/22 

460 

2370  gms. 

79  cc.4  culture  filtrate  3421  by  mouth 

Remained  healthy 
Released  11/14/22 

'Approximately  1  cc.  of  toxin  to  240  grams  live  weight. 
Approximately  1  cc.  of  toxin  to  120  grams  live  weight. 
"Approximately  1  cc.  of  toxin  to  60  grams  live  weight. 
Approximately  1  en.  of  toxin  to  30  grams  live  weight;. 

of  their  body  weight.  Occasionally  other  chickens  have  succumbed  fol- 
lowing the  ingestion  of  smaller  amounts  of  the  culture.  The  resist- 
ance which  some  healthy  chickens  possess  is  shown  by  the  data  in 
Table  9.  The  tolerance  of  healthy  chickens  to  both  the  culture  and  the 
culture  nitrate  under  experimental  conditions,  together  with  the  fact 
that  the  course  of  the  disease  is  more  rapidly  fatal  in  experimental 
chickens  affected  with  contagious  epithelioma,  suggests  the  possible 
part  played  by  secondary  or  predisposing  causes. 

In  feeding  C.  botulinum  type  C  (3421)  to  horses,  it  was  found 
that  50  grams  of  the  culture  per  1000  pounds  live  weight  was  sufficient 
to  induce  a  transitory  illness  (see  Table  10).  One  horse  given  100 
grams  per  1000  pounds  live  weight,  developed  symptoms  of  weak- 
ness, paralysis,  obstinate  constipation,  and  incoordination  of  move- 
ment, followed  by  decumbency  on  the  fourth  day.  In  the  decumbent 
position  the  feet  were  spasmodically  moved  in  a  running  motion, 
while  the  animal  remained  nervously  alert  and  conscious  of  the  sur- 
roundings. Death  followed  on  the  eighth  day. 

Three  out  of  four  cattle,  weighing  from  240  to  985  pounds  each, 
which  were  fed  from  100  to  400  grams,  per  1000  pounds  live  weight, 
of  C.  botulinum  type  C,  culture  3421,  showed  marked  symptoms  of 
incoordination,  dulness,  and  stiffness  (see  Table  10).  The  posterior 
limbs  were  unsteady,  the  animals  falling  on  their  knees  if  urged  to 
walk  rapidly.  After  exertion  the  animals  appeared  nervous  and 
excitable,  but  following  rest  they  resumed  a  more  normal  attitude. 
The  symptoms  gradually  subsided  in  a  few  days.  The  daily  tempera- 


20 


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ture  of  the  animals  did  not  show  any  appreciable  elevation  during  the 
course  of  the  illness.  One  cow  (497)  given  200  grams  of  C.  botulinum 
type  C,  culture  3421,  per  1000  pounds  live  weight,  remained  unaffected. 

The  results  of  several  experiments  indicate  a  resistance  of  sows 
and  pigs  to  C.  botulinum  type  C.  Nursing  pigs  twenty-four  days 
old  were  fed  .1  cc.,  .2  cc.,  .4  cc.,  .8  cc.,  and  1  cc.,  per  pound  live  weight, 
of  culture  3421  with  no  apparent  effect  other  than  a  slight  elevation 
of  the  rectal  temperatures  between  the  .fourth  and  sixth  days  (see 
Table  11).  Nursing  pigs  thirty  to  thirty-four  days  old  which  were 
fed  1  cc.,  1.5  cc.,  and  2  cc.,  per  pound  live  weight,  of  C.  botulinum 
type  C,  culture  3421,  also  showed  no  illness  (see  Table  12).  Massive 
amounts  of  C.  botulinum  type  C  culture  were  fed  to  sows  at  the  time 
young  pigs  were  being  suckled,  but  without  noticeable  effect  in  either 
the  sow  or  the  pigs  (see  Table  13). 

Bacteriologic  examination  of  soil  samples  obtained  several  months 
later  from  the  chicken  yard  from  which  specimens  3421,  3422,  and 
3423  were  received,  proved  negative  to  C.  botulinum  type  C. 


SPECIMENS  3466,  3467,  AND  3468 

Three  chickens,  Nos.  3466,  3467,  and  3468,  were  received  for  ex- 
amination on  July  20,  1922,  from  Mr.  I.  L.  Durbin,  of  Taylorville, 
Christian  county,  Illinois.  Fifteen  half-grown  chickens  had  died  in 


FIG.  8. — CHICKENS  3466  AND  3467.     TYPICAL  SPONTANEOUS 
CASES  OF  TYPE  C  BOTULINUS  POISONING 

this  flock.  The  clinical  symptoms  displayed  by  the  three  chickens  on 
their  arrival  consisted  of  weakness  and  diarrhea.  The  eyes  were 
closed,  and  the  chickens  seemed  to  be  asleep  (see  Fig.  8).  One  of 
them  (3468)  appeared  during  the  examination  to  be  greatly  im- 
proved over  its  previous  condition  as  described  by  the  owner  and 
was  returned  to  the  flock.  Chickens  3466  and  3467  were  killed  for 
autopsy.  No  gross  lesions  were  encountered. 


24 


BULLETIN  No.  246 


[October, 


Anaerobic  cultures  of  the  intestinal  content  and  spleen  tissue  of 
chicken  3466  yielded  toxic  cultures.     The  purification  of  this  strain 


FIG.  9. — CHICKEN  554,  SHOWING  CLINICAL  ASPECTS  OF  BOTULINUS  POISONING, 
TYPE  C,  EXPERIMENTALLY  INDUCED  BY  FEEDING  (See  Table  15) 

by  plate  and  deep-tube  cultures  was  not  accomplished.    Guinea  pigs, 
however,  which  were  fed  .5  cc.  of  the  toxin  elaborated  in  impure 

TABLE  12. — RESISTANCE  OF  PIGS  TO  MASSIVE  AMOUNTS  OF  TYPE  C  CULTURE 
3421  GIVEN  BY  MOUTH 


Identification 

Treatment,  November  13,  1922 

Results 

Pig  No. 

Sex 

Age 

Weight 

534 

2 

M 

30  days 

81bs. 

8  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

535 
3 

M 

30  days 

91bs. 

18  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

536 
4 

F 

30  days 

10  Ibs. 

Control 

Remained  healthy 
Released  12/1/22 

537 
5 

F 

30  days 

9^  Ibs. 

Control 

Remained  healthy 
Released  12/1/22 

538 
6 

M 

30  days 

9^  Ibs. 

Control 

Remained  healthy 
Released  12/1/22 

539 
7 

F 

30  days 

11  Ibs. 

11  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

540 
8 

F 

30  days 

6  Ibs. 

12  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

541 
1 

M 

34  days 

15  Ibs. 

15  oc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

542 
2 

F 

34  days 

14  Ibs. 

21  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

543 
3 

F 

34  days 

14  Ibs. 

Control 

Remained  healthy 
Released  12/1/22 

544 
4 

M 

34  days 

18  Ibs. 

Control 

Remained  healthy 
Released  12/1/22 

545 
6 

M 

34  days 

6  Ibs. 

6  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

546 
7 

M 

34  days 

12  Ibs. 

18  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  12/1/22 

1923] 


CLOSTRIDIUM  BOTULINUM  TYPE  C 


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26 


BULLETIN  No.  246 


[October, 


TABLE  13. — RESISTANCE  OF  Sows  AND  NURSING  PIGS  TO  TYPE  C  CULTURE  3421 

FED  TO  Sow 


Identification 

Treatment,  November  8,  1922 

Results 

No. 

Animal 

Sex 

Age 

Weight 

506 
OPC 

Sow 

F 

2  yrs. 

450  Ibs. 

150  cc.  culture  3421  by  mouth 

Remained  healthy 
Released  11  /2S/22 

681 
1 

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of 
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M 

12  days 

91bs. 

None 

Remained  healthy 
Released  11/23/22 

682 
2 

M 

12  days 

gibs. 

None 

Remained  healthy 
Released  11/23/22 

683 
3 

F 

12  days 

91bs. 

None 

Remained  healthy 
Released  11/23/22 

684 
4 

F 

12  days 

9H  Ibs. 

None 

Remained  healthy 
Released  11/23/22 

685 
5 

F 

12  days 

10  Ibs. 

None 

Remained  healthy 
Released  11/23/22 

686 
6 

F 

12  days 

10  Ibs. 

None 

Remained  healthy 
Released  11/23/22 

687 
7 

F 

12  days 

9  Ibs. 

None 

Remained  healthy 
Released  11/23/22 

TABLE  14. — PROTECTIVE  CHARACTER  OF  BOTULINUS  ANTITOXIN  TYPE  C1  TO 
CULTURE  FILTRATE  3466 


Identification 

Treatment,  August   5,  1922 

Results 

Guinea 
& 

Weight 

662 

350  gms. 

.5  cc.  culture  filtrate  3466  by 
mouth 

.5  cc.  botulinus  antitoxin  type  C, 
296  subcutaneously 

Died  8/6/22, 
8  a.m. 

687 

350  gms. 

.5  cc.  culture  filtrate  3466  by 
mouth 

1  cc.  botulinus  antitoxin  type  C, 
296,  subcutaneously 

Remained  healthy 
Released  9/1/22 

753 

400  gms. 

.  5  cc.  culture  filtrate  3466  by 
mouth 

1.5  cc.  botulinus  antitoxin  type 
C,  296,  subcutaneously 

Remained  healthy 
Released  9/1/22 

253 

400  gms. 

.  5  cc.  culture  filtrate  3466  by 
mouth 

Control 

Died  8/6/22, 
8  a.m. 

'Furnished  by  Dr.  G.  W.  McCoy,  Director  of  Hygienic  Laboratory,  Washington,  D.  C. 


1923] 


CLOSTRIDIUM  BOTULINUM  TYPE  C 


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BULLETIN  No.  246 


[October, 


TABLE  15. — RESISTANCE  OF  CHICKENS  FED  MASSIVE  AMOUNTS  OF  TYPE  C 

CULTURE  3466 » 


Identification 

Treatment,  November  14,  1922 

Results 

Chicken 
No. 

Weight 

549 

2460  gms. 

10  cc 

2  meat-mash  culture  3466  by  mouth 

Remained  healthy 
Released  12/12/22 

550 

1280  gms. 

10  cc 

*  meat-mash  culture  3466  by  mouth 

Remained  healthy 
Released  12/12/22 

551 

2320  gms. 

38  cc 

4  meat-mash  culture  3466  by  mouth 

Remained  healthy 
Released  12/12/22 

552 

1810  gms. 

30  cc 

4  meat-mash  culture  3466  by  mouth 

Leg  weakness  and  prostra- 
tion 11/18    Died  11/24/22 

553 

2130  gms. 

71  cc 

6  meat-mash  culture  3466  by  mouth 

Remained  healthy 
Released  12/12/22 

554« 

2130  gms. 

71  cc. 

5  meat-mash  culture  3466  by  mouth 

Leg  weakness  11/15 
Died  11/16/22 

•Culture  was  impure,  containing  a  spore-forming  rod  resembling  B.  sporogenes. 
'Approximately  1  gram  of  m.m.  culture  to  240  grams  live  weight. 
'Approximately  1  gram  of  m.m.  culture  to  120  grams  live  weight 
Approximately   1  gram  of  m.m.  culture  to  60  grams  live  weight. 
'Approximately  1  gram  of  m.m.  culture  to  30  grams  live  weight. 
•This  chicken  showed  slight  symptoms  of  ocular  roup  at  the  time  of  treatment. 

cultures  were  protected  by  injections  of  1  and  1.5  cc.  of  type  C  anti- 
toxin (see  Table  14).  The  toxin  elaborated  in  impure  cultures  of 
anaerobe  3466  was  also  fed  in  varying  amounts  to  six  chickens.  Two 
of  these  developed  typical  symptoms  of  botulinus  poisoning  and  died. 
(See  Table  15  and  Fig.  9). 

Cultures  of  soil  samples  obtained  several  months  later  from  the 
chicken  yard  of  the  premises  where  chicken  3466  had  been  kept, 
proved  positive  to  C.  botulinum  type  C  as  determined  by  toxin- 
antitoxin  tests  on  guinea  pigs,  using  unpurified  cultures  and  culture 
filtrates. 

BOTULINUS  ANTITOXIN  TYPE  C 

An  antitoxin  has  been  prepared  from  goats  and  calves  by  giving  re- 
peated injections  subcutaneously  of  non-lethal  amounts  of  the  culture 
filtrate  of  type  C  strains  isolated  from  chickens.  The  specific  pro- 
tective character  of  this  antitoxin  has  been  consistently  demonstrated 
in  guinea  pigs.  The  antitoxin  of  the  homologous  type  C  strain  pro- 
tects against  the  toxin  of  the  two  other  strains  isolated  from  sporadic 
cases  of  leg  weakness  and  limberneck  in  poultry  described  in  this  bulle- 
tin, as  well  as  against  the  toxin  of  a  strain1  isolated  from  the  stomach 


1  Since  this  report  was  written,  Dr.  H.  H.  Seely  of  White  Hall,  Illinois,  has 
submitted  to  the  Laboratory  of  Animal  Pathology  and  Hygiene  for  bacteriologic 
examination  a  sample  of  stomach  contents  (526)  from  a  horse,  following  death. 
An  anaerobic  toxin-producing,  spore-forming  rod  was  isolated  from  this  specimen 
by  seeding  dilutions  of  the  original  sample  in  glucose  agar  tubes  and  incubating 


CLOSTRIDIUM  BOTULINUM  TYPE  C 


29 


contents  of  a  horse  and  one1  from  the  intestinal  contents  of  a  steer, 
the  type  C  strain  of  Bengtson,  and  the  parabotulinus  organism  of 
Seddon  (see  Table  16).  The  antitoxin  prepared  against  the  fly-larvae 
strain  of  Bengtson  protects  against  the  toxin  elaborated  by  the  three 
chicken  strains,  the  type  C  strains  from  a  horse  and  a  steer,  as  well  as 
against  the  parabotulinus  toxin  of  Seddon.  The  type  C  antitoxin 
prepared  against  the  toxin  of  chicken  strains  has  also  been  used  to 
determine  the  presence  of  type  C  strains  in  cultures  from  two  samples 
of  soil  from  poultry  yards,  as  well  as  in  certain  fly  larvae  received  from 
Dr.  E.  W.  Saunders,  of  St.  Louis,  Missouri.  The  prophylactic  value 
of  the  botulinus  antitoxin  type  C  in  healthy  animals  is  suggested  in 
experimental  results,  but  there  is  as  yet  but  limited  field  evidence 


at  37°  C.  Single  isolated  colonies  proved  to  be  similar,  culturally  and  morpho- 
logically, to  C.  botulinum  type  C.  The  identity  of  the  anaerobe  was  finally  deter- 
mined by  positive  immunologic  tests,  reported  in  the  following  table. 

TABLE  A. — PROTECTIVE  CHARACTER  OF  TYPE  C  ANTITOXIN  3421  AGAINST  TOXIN  526 


Identification 

Treatment,   December   11,   1922 

Results 

Guinea  pig 

No. 

Weight 

1467 

250-300    gms. 

100    units    Botulinus    antitoxin   type    A 
5    cc.    culture    filtrate    526    by    mouth 

Died  12/12/22,    3 

p.m. 

1465 

250-300   gms. 

100    units    Botulinus    antitoxin    type    B 
5    cc.    culture    filtrate    526    by    mouth 

Died  12/12/22,    10  a.m. 

1681 

250-300    gms. 

1  cc.  BotuTnus  antitoxin  type  C  (3421) 
.5    cc.    culture    filtrate    526    by   mouth 

Remained    healthv 
Released   1/30/23 

Symptoms. — The  symptoms  observed  in  the  spontaneously  affected  horses  con- 
sisted of  a  period  of  stupor  of  ten  to  twelve  hours,  followed  by  decumbency.  Evi- 
dence of  paralysis  or  weakness  of  the  posterior  quarters  was  noticeable  when  the 
affected  animals  attempted  to  rise.  Irritability  and  hypersensitiveness,  together 
with  accelerated  respiration,  preceded  death.  After  lying  down,  animals  survived 
from  twelve  hours  to  several  days.  The  temperature  ranged  fom  98.8°  F.  to 
101.8°  F. ;  pulse,  weak,  54  to  65 ;  respiration  16  to  20.  Four  other  horses  in  this 
herd  died  during  a  period  of  twelve  days. 

Feed. — The  horses  had  been  fed  over-ripe  clover  hay,  timothy  hay,  and  corn. 
They  had  also  had  access  to  pasture.  Samples  of  the  feeds  submitted  were  tested 
for  the  presence  of  toxin,  with  negative  results.  No  evidence  of  the  presence  of 
toxin-producing  anaerobes  in  the  feed  was  obtained  by  cultural  methods. 

Soil. — Samples  of  soil  from  the  yard  where  the  horses  died  yielded  cultures 
which  proved  toxic  for  guinea  pigs  when  given  by  the  mouth.  The  intoxication 
was  offset  only  by  injecting  type  C  antitoxin.  The  soil  culture  was  not  obtained 
free  from  contamination  either  by  seeding  in  glucose-agar  tubes  or  by  plating. 

The  distribution  of  C.  ~botulinum  in  nature  forbids  a  diagnosis  of  botulism  on 
the  basis  of  bacteriologic  findings  alone.  Symptoms  in  spontaneously  affected 
horses,  together  with  evidence  of  the  presence  of  C.  botulinum  in  the  digestive 
tube  of  fatally  afflicted  animals  and  its  presence  in  the  soil  of  the  premises  on 
which  the  horses  died,  are  worthy  of  consideration  in  investigational  studies  look- 
ing to  the  establishment  of  the  causative  agent  of  similar  diseases  of  horses,  of 
unknown  etiology. 

1  On  January  2,  1923,  Dr.  H.  E.  Rinehart  of  Bushville,  Illinois,  submitted  kid- 
neys (780),  lung  (781),  heart  (782),  trachea  (783),  and  intestinal  content  (784) 
of  a  three-year-old  steer  that  had  died  after  an  illness  of  twenty-four  hours.  Two 
other  steers  in  a  herd  of  113  in  the  same  feed  lot  had  died  from  an  acute  disease. 
Petechial  and  ecchymotic  hemorrhages  were  very  noticeable  on  the  heart,  lung, 
and  serous  covering  of  the  intestines  received  for  examination. 


30 


BULLETIN  No.  246 


[October, 


TABLE  16. — PROTECTIVE  CHARACTER  OF  TYPE  C  ANTITOXIN  3421  AGAINST 
HOMOLOGOUS  AND  HETEROLOGOUS  TOXIN 


Guinea 
pig 

No. 

Treatment 

Results 

Date 

Toxin,  by  mouth 

Type  C  antitoxin 
3421, 
subcutaneously 

659 

9/25/22 

.5  cc.  avian  strain  culture  filtrate  3421 

2  cc.  goat  antitoxin 

Remained  healthy 

654 

P  ,'25/22 

.5  cc.  avian  strain  culture  filtrate  3421 

None 

Died  9/26/22 

671 

9/25/22 

.5  cc.  avian  strain  culture  filtrate  3420 

2  cc.  goat  antitoxin 

Remained  healthy 

799 

9/25/22 

.  5  cc.  avian  strain  culture  filtrate  3420 

None 

Died  9/26/22 

264 

9/25/22 

.  5  cc.  fly  larvae  strain  culture  filtrate  2070, 
Bengtson 

2  cc.  goat  antitoxin 

Remained  healthy 

726 

9/25/22 

.  5  cc.  fly  larvae  strain  culture  filtrate  2070, 
Bengtson 

None 

Died  9/26/22 

1308 

1/19/23 

.  5  cc.  avian  strain  culture  filtrate  3466 

2  cc.  goat  antitoxin 

Remained  healthy 

1865 

1/19/23 

.  5  cc.  avian  strain  culture  filtrate  3466 

None 

Died  1/21/23 

1259 

11/26/22 

.  5  cc.  avian  strain  culture  filtrate  487 

2  cc.  goat  antitoxin 

Remained  healthy 

1274 

11/26/22 

.  5  cc.  avian  strain  culture  filtrate  487 

None 

Died  11/27/22 

1681 

12/11/22 

.  5  cc.  equine  strain  culture  filtrate  526 

2  cc.  calf  antitoxin 

Remained  healthy 

1465 

12/11/22 

.5  cc.  equine  strain  culture  filtrate  526 

None 

Died  12/12/22 

1853 

1/23/23 

.  5  cc.  bovine  strain  culture  filtrate  784 

2  cc.  calf  antitoxin 

Remained  healthy 

1502 

1/23/23 

.  5  cc.  bovine  strain  culture  filtrate  784 

None 

Died  1/24/23 

947 

7/6/23 

.5  cc.  parabotulinus  strain  culture  filtrate, 
Seddon 

2  cc.  caif  antitoxin 

Remained  healthy 

935 

7/6/23 

.5  cc.  parabotulinus  strain  culture  filtrate, 
Seddon 

None 

Died   7/8/23 

A  composite  sample  of  kidney,  lung,  and  heart  was  injected  subcutaneously 
into  a  healthy  rabbit.  Six  days  later  the  rabbit  was  found  dead.  Direct  cultures 
from  the  heart's  blood  of  the  rabbit  yielded  a  small,  Gram-negative  rod  indis- 
tinguishable from  Pasteurella  bovisepticum.  Cultures  of  the  intestinal  content 
(784')  of  the  steer  in  meat -mash  media  proved  fatal  to  guinea  pigs  when  given 
by  the  mouth.  The  symptoms  in  guinea  pigs  were  similar  to  the  symptoms  of 
botulism.  Single  colonies  in  glucose  agar  were  subcultured  and  purified.  The 
results,  which  are  shown  in  the  following  table,  suggest  its  close  relation  to 
C.  botulinum  type  C. 

TABLE  B. — PROTECTIVE  CHARACTER  OF  TTPE  C  ANFITOXIN  3421  AGAI.VST  TOXIN  784 


Identification 

Treatment,  January  19,   1923 

Results 

Guinea  pig 
No. 

Weight 

1651 

250-300   gms. 

100  units  Botulinus  antitoxin  type  A 
.5  cc.  culture  filtrate  784  by  mouth 

Died   1/22/23 

1159 

250-300   gms. 

100  units  Botulinus  antitoxin  type  B 
.5  cc.  culture  filtrate  784  by  mouth 

Died    1/21/23 

1652 

250-300  gms. 

1  cc.  Botulinus  antitoxin  type  C  (3421) 
5  cc.  culture  filtrate  784  by  mouth 

Remained  healthy 
Released    1/30/23 

The  widespread  distribution  of  C.  botulinum  type  C  in  nature  may  be  the 
explanation  of  the  positive  findings  in  the  intestinal  tube  of  the  steer.  In  view 
of  the  susceptibility  of  cattle  to  type  C  poisoning  under  experimental  conditions, 
the  possible  relation  of  this  organism  to  acute  paralytic  diseases  of  cattle  may 
be  worthy  of  further  study. 


1923] 


CLOSTEIDIUM  BOTULINUM  TYPE  C 


31 


obtained  under  conditions  properly  controlled  to  justify  the  use  of 
the  antitoxin  in  the  prevention  of  type  C  poisoning  in  animals. 

It  has  been  observed  that  the  subcutaneous  injection  of  type  C 
toxin  (filtered  cultures)  is  more  fatal  to  goats  and  sheep  than  is  the 
toxin  of  type  A  or  B,  and  that  these  animals,  like  yearling  calves, 
may  succumb  even  after  they  have  withstood  several  injections  of 
toxin.  Sheep  appear  to  be  even  less  resistant  to  subcutaneous  injec- 
tions of  the  filtered  toxin  than  goats  and  calves. 

The  sterile  filtrate  of  cultures  used  in  hyper-immunizing  animals 
in  the  preparation  of  antitoxin  has  proven  labile,  the  toxicity  of  the 
filtrate  decreasing  rapidly  upon  exposure  at  room  temperature. 

THE  DEVELOPMENT  OF  TYPE  C  TOXIN  IN  SHELLED  CORN 

The  tolerance  exhibited  by  chickens,  horses,  and  cattle  following 
the  ingestion  of  toxin  developed  in  meat-mash  medium  led  to  experi- 
ments designed  to  determine  whether  toxin  may  not  develop  in  grains 
and  hays,  and  if  so  whether  such  toxin  is  as  potent  as  that  elaborated 


TABLE  17. — DEVELOPMENT  OF  TOXIN  IN  SHELLED  CORN  INOCULATED  WITH 

TYPE  C  TOXIN-FREE  SPORES  AND  ITS  FAILURE  TO  DEVELOP  IN 

SAMPLES  OF  OTHER  FEEDS  AND  SOIL 


Identification 

Inoculum, 
3/14/23 
TypeC 
spores,  3421 

Treatment  of  guinea  pigs 

Sample 
No. 

-   Sterile 

Non-sterile 

Culture  given  by 
mouth  6/28/23 

Results 

1658 

Soil 

10,000,000 

.5cc. 

Negative 

1659 

Soil 

10,000,000 

.5cc. 

Negative 

1660 

Shelled  corn 

10,000,000 

.5cc. 

Typical  fatal  symp- 
toms of  botulism 

1661 

Shelled  corn 

10,000,000 

.5cc. 

Negative 

-    1662 

Corn  meal 

10,000,000 

.5cc. 

Negative 

1663 

Corn  meal 

10,000,000 

.5cc. 

Negative 

1664 

Oats 

10,000,000 

.5cc. 

Negative 

1665 

Data 

10,000,000 

.5cc. 

Negative 

1666 

Ground  alfalfa 

10,000,000 

.5cc. 

Negative 

1667 

Ground  alfalfa 

10,000,000 

.5cc. 

Negative 

1668 

Ground  timothy 

10,000,000 

.5cc. 

Negative 

1669 

Ground  timothy 

10,000,000 

.Sec. 

Negative 

1670 

Tankage 

10,000,000 

.5cc. 

Negative 

1671 

Tankage 

10,000,000 

.5cc. 

Negative 

1672 

Silage 

10,000,000 

.5cc. 

Negative 

1673 

Silage 

10,000,000 

.5cc. 

Negative 

32  BULLETIN  No.  246  [October, 

in  meat  medium.  The  possibility  of  feeds  becoming  contaminated  was 
further  suggested  by  the  distribution  of  the  spores  in  nature. 

Duplicate  series  of  samples  of  soil,  shelled  yellow  corn,  corn  meal, 
oats,  ground  alfalfa,  ground  timothy,  tankage,  and  silage  placed  in 
eight-ounce  clear  glass  bottles,  were  inoculated  with  10  million  type  C 
spores  (3421)  each  (see  Table  17).  Previous  to  inoculation  one  of 
each  of  the  duplicate  samples  was  sterilized  by  fractional  sterilization 
and  cultured  to  determine  sterility. 

The  spores  were  washed  ten  times  and  were  heated  at  70°  C.  for 
five  minutes.  The  non-toxic  character  of  the  spore  suspension  was 
established  in  guinea  pigs,  while  subcultures  showed  that  the  vitality 
of  the  spores  was  noticeably  reduced.  After  allowing  all  the  inocu- 
lated samples  to  incubate  at  room  temperature  in  a  dark  place  for  one 
hundred  and  thirty-six  days,  50  cc.  of  sterile  saline  was  added  to  each 
sample  and  mechanically  shaken  for  five  minutes.  The  liquor  from 
each  bottle  was  then  fed  in  amounts  of  .5  cc.  to  guinea  pigs.  That 
from  shelled  corn  (1660)  was  the  only  one  of  the  series  which  proved 
toxic  (see  Table  17).  When  injected  subcutaneously,  .0001  cc.  of  the 
liquor  proved  fatal  to  guinea  pigs.  The  intoxication  induced  by 
subcutaneous  injection  and  also  that  induced  by  feeding  was  offset 
by  type  C  antitoxin. 

In  other  series  of  samples  of  grains  and  hays  which  have  been 
inoculated  with  toxin-free  spores  of  type  C,  consistent  growth  has  not 
been  obtained,  but  the  results  suggest  that  under  favorable  conditions 
type  C  toxin  may  develop  in  shelled  corn  and  possibly  in  other  feeds. 


SUMMARY 

1.  A  disease  of  chickens  and  ducks  characterized  by  the  sudden 
development  of  lameness  or  leg  weakness,  diarrhea,  ptosis,  prostra- 
tion, and  limberneck,  analogous  to  avian  botulism  type  A  and  fol- 
lowed in  some  cases  by  recovery  but  in  many  others  by  death  from 
respiratory  paralysis,  has  been  encountered  in  five  flocks  in  Illinois. 

2.  The  results  of  toxicogenic  tests  and  bacteriologic  findings  of 
the  intestinal  contents  and  internal  organs  of  affected  chickens  sug- 
gest that  one  type  of  the  avian  botulism  syndrome  is  closely  associated 
with  the  ingcstion  of  a  bacterial  toxin  related  to,  if  not  identical  with, 
the  toxin  of  the  anaerobe  isolated  from  the  larvae  of  Lucilia  caesar 
by  Bengtson  of  the  Hygienic  Laboratory,  who  designated  it  Bacillus 
botulinus  type  C,  or  Clostridium  botulinum  type  C. 

3.  The  toxic  anaerobe  isolated  from  affected  chickens  is  also  closely 
related  to  the  parabotulinus  organism  isolated  by  Seddon  of  the  Vet- 
erinary Institute,  Melbourne  University,  from  cases  of  bulbar  paraly- 
sis of  cattle.     The  antitoxin  prepared  from  the  avian  type  C  strains 


1923}  CLOSTRIDIUM   BOTULINUM   TYPE  C  33 

protects  guinea  pigs  against  lethal  doses  of  the  parabotulinus  toxin, 
as  well  as  against  similar  toxic  anaerobes  isolated  from  the  stomach 
contents  of  a  spontaneously  affected  horse  and  the  intestinal  contents 
of  an  affected  steer  in  two  outbreaks  in  Illinois. 

4.  The  symptoms  of  type  C  botulinus  poisoning  in  guinea  pigs  are 
similar  to  the  symptoms  of  type  A  and  type  B  botulinus  poisoning, 
yet  neither  type  A  nor  type  B  botulinus  antitoxin  affords  protection 
against  the  type  C  poisoning.    In  horses,  also,  type  C  poisoning  re- 
sembles types  A  and  B  poisoning.    Since  chickens  are  susceptible  to 
type  A  toxin  as  well  as  to  type  C,  a  diagnosis  of  the  causative  agent  of 
limberneck-like  diseases  may  be  expedited  in  the  laboratory  by  im- 
munologic  tests. 

5.  C.  botulinum  type  C  grows  less  vigorously  than  type  A  or  B 
in  media  employed  in  the  isolation  of  those  strains.     Single  colony 
subcultures  of  type  C  may  sometimes  fail  to  grow  or  for  some  reason 
prove  non-lethal.     The  spores  are  less  resistant  to  heat  than  are  the 
spores  of  types  A  and  B. 

6.  The  resistance  of  certain  chickens  to  type  C  toxin  has  been 
suggested  by  the  low  mortality  in  some  affected  flocks  as  well  as  by 
the  tolerance  of  healthy  chickens  to  the  culture  and  culture  filtrate 

.under  experimental  conditions.  Chickens  of  low  vitality,  suffering 
from  contagious  epithelioma,  have  been  shown  to  be  more  susceptible 
to  type  C  toxin  than  are  healthy  fowls.  The  exact  significance  of  sec- 
ondary factors  such  as  parasitism  and  contagious  epithelioma,  which 
might  lower  the  vitality  of  chickens,  independent  of  the  important 
factor  of  the  quantity  of  toxin  ingested,  probably  merits  further 
investigation. 

7.  It  has  been  shown  that  horses,  cattle,  and  sheep  are  susceptible 
to  the  subcutaneous  injection  of  C.  botulinum  type  C.    Small  amounts 
in  the  feed  of  horses  and  cattle  daily  for  a  period  of  ten  consecutive 
days  failed  to  induce  symptoms.     Single  massive  amounts  given  by 
the  mouth,  however,  induced  illness  in  cattle  and  death  in  horses. 

8.  Sows,  nursing  pigs,  shotes,  and  dogs  have  been  shown  to  be 
resistant  to  the  culture  and  culture  nitrate  of  C.  botulinum  type  C 
when  given  in  the  feed. 

9.  An  antitoxin  has  been  prepared  from  goats  and  calves  by  re- 
peated injection  of  the  culture  filtrate  of  C.  botulinum  type  C.    The 
antitoxin  is  of  prophylactic  value  in  guinea  pigs;    it  is  also  useful 
in  identifying  the  toxin  in  immunologic  tests  where  the  type  C  botuli- 
nus organism  is  suspected.     Sufficient  evidence  has  not  as  yet  been 
obtained  to  show  that  the  antitoxin  is  of  value  in  the  treatment  or 
prevention  of  type  C  poisoning  occurring  in  poultry  or  other  animals 
under  farm  conditions. 


34  BULLETIN  No.  246 

10.  The  presence  of  C.  botuUnum  type  C  in  the  soil  of  two  poultry 
yards  several  months  after  manifest  symptoms  of  the  disease  in 
chickens  occurred  on  the  premises,  as  well  as  in  the  soil  of  a  barn- 
yard where  horses  had  died  of  a  paralytic  disease,  may  be  significant 
of  the  distribution  of  this  organism  in  nature. 

11.  The  toxin-free  spores  of  C.  botuUnum  type  C  are  capable  of 
elaborating  a  highly  lethal  toxin  in  shelled  corn  sealed  in  glass  bottles 
and  kept  at  room  temperature.  The  development  of  the  toxin  in  all 
corn  samples  inoculated  was  not  consistent  under  laboratory  conditions. 


UNIVERSITY  OF  ILLINOIS-URBANA 


